The present invention relates to a novel starch branching enzyme of potato. More specifically, the present invention relates to an amino acid sequence of a second starch branching enzyme (SBE II) of potato and a fragment thereof as well as their corresponding DNA sequences. Furthermore, the invention relates to vectors comprising such DNA sequences, to processes for production of transgenic potatoes, and to the use of said potatoes for the production of starch.
Starch is a complex mixture of different molecule forms differing in degree of polymerization and branching of the glucose chains. Starch consists of amylose and amylopectin, whereby the amylose consists of an essentially linear xcex1-1,4-glucan and amylopectin consists of xcex1-1,4-glucans connected to each other via xcex1-1,6-linkages and, thus, forming a branched polyglucan. Thus, starch is not a uniform raw material.
Starch is synthesized via at least three enzymatic reactions in which ADP glucose phosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21) and starch branching enzyme (EC 2.4.1.18) are involved. Starch branching enzyme (SBE, also called Q-enzyme) is believed to have two different enzymatic activities. It catalyzes both the hydrolysis of xcex1-1,4-glucosidic bonds and the formation of xcex1-1,6-glucosidic bonds during synthesis of the branched component in starch, i.e. amylopectin.
Plant starch is a valuable source of renewable raw material used in, for example, the chemical industry (Visser and Jacobsen, 1993). However, the quality of the starch has to meet the demands of the processing industry wherein uniformity of structure is an important criterion. For industrial application there is a need of plants only containing amylose starch and plants only containing amylopectin starch, respectively.
Processes for altering the amylose/amylopectin ratio in starch have already been proposed. For example, in WO95/04826 there is described DNA sequences encoding debranching enzymes with the ability to reduce or increase the degree of branching of amylopectin in transgenic plants, e.g. potatoes.
In WO92/14827 plasmids are described having DNA sequences that after insertion into the genome of the plants cause changes in the carbohydrate concentration and the carbohydrate composition in regenerated plants. These changes can be obtained from a sequence of a branching enzyme that is located on these plasmids. This branching enzyme is proposed to alter the amylose/amylopectin ratio in starch of the plants, especially in commercially used plants.
WO92/14827 describes the only hitherto known starch branching enzyme in potato and within the art it is not known whether other starch branching enzymes are involved in the synthesis of branched starch of potato.
In Mol Gen Genet (1991) 225:289-296(Visser et al., there is described inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Inhibition of the enzyme in potato tuber starch was up to 100% in which case amylose-free starch was provided.
However, the prior known methods for inhibiting amylopectin have not been that successful and, therefore, alternative methods for inhibiting amylopectin are still highly desirable (Mxc3xcller-Rxc3x6ber and Koxcex2mann, 1994; Martin and Smith, 1995).
The object of the present invention is to enable altering the degree of amylopectin branching and the amylopectin/amylose ratio in potato starch.
According to the present invention this object is achieved by providing a novel isolated DNA sequence encoding a second starch branching enzyme, SBE II, and fragments thereof, which after insertion into the genome of the plants cause changes in said branching degree and ratio in regenerated plants.
Within the scope of the present invention there is also included the amino acid sequence of SBE II and fragments thereof.
Also variants of the above DNA sequence resulting from the degeneracy of the genetic code are encompassed.
The novel DNA sequence encoding SBEII, comprising 3074 nucleotides, as well as the corresponding amino acid sequence comprising 878 amino acids, are shown in SEQ ID No. 1. One 1393 nucleotides long fragment of the above DNA sequence, corresponding to nucleotides 1007 to 2399 of the DNA sequence in SEQ ID No. 1, as well as the corresponding amino acid sequence comprising 464 amino acids, are shown in SEQ ID No. 2.
Furthermore, there are provided vectors comprising said isolated DNA-sequences and regulatory elements active in potato. The DNA sequences may be inserted in the sense or antisense (reversed) orientation in the vectors in relation to a promoter immediately upstream from the DNA sequence.
Also there is provided a process for the production of transgenic potatoes with a reduced degree of branching of amylopectin starch, comprising the following steps:
a) transfer and incorporation of a vector according to the invention into the genome of a potato cell, and
b) regeneration of intact, whole plants from the transformed cells.
Finally, the invention provides the use of said transgenic potatoes for the production of starch.
The invention will be described in more detail below in association with an experimental part and the accompanying drawings.